The quest for peripheral tolerance in human transplantation remains unfulfilled and new insight is required. Therapeutic strategies able to induce peripheral tolerance in murine models have thus far been unable to reliably induce tolerance in non-human primates (NHP). Nonetheless, murine models have been instructive in defining important targets and demonstrating that combination therapy is much more robust. Indeed, anti-CD154, even as a single agent allowed significant treatment-free survival in NHP, even if true tolerance was not achieved. Based on potent synergy with anti-CD154 and other agents in the most stringent murine transplant models and a unique mechanisms of action that include induction of the endogenous inhibitory costimulatory molecule, CTLA-4 and de novo generation of regulatory T cells (Tregs), we now propose to study the ability of anti-CD45RB-based regimens to induce renal allograft tolerance in NHP. Preliminary findings using an anti-human CD45RB as a single agent or with Rapamycin demonstrate occasional long-term survival. However, success may be hampered by rapid development of anti-murine Abs (MAMA). Based on potent synergy both in terms of tolerance induction and in terms of mechanisms of action, we now propose: In Aim 1, we will determine the efficacy of anti-CD45RB plus anti-CD40 (blockade of positive costimulation through CD40/CD154 combined with augmentation of negative costimulatory signaling through CTLA-4);In Aim 2, combining anti-CD45RB with sCD83, a novel and powerful inhibitor of DC maturation (since tolerogenic DC combined with de novo induction of Treg by anti-CD45RB forms a positive feedback loop for generating Tregs). Additional agents such as donor-specific transfusion (DST) or Rapamycin will be added if necessary, again based on potent synergy in both murine and NHP models. In Aim 3, mechanistic studies will be performed to extend insight gained from murine studies, and specifically address the effects of the added agents on Treg generation, anergy, deletion, and inhibition of B cell and DC activation. In Aim 4, specific signals that induce de novo conversion of non-regulatory cells into regulatory T cells (Tregs) without requiring cell activation will be defined. This will provide new insight into Treg immunobiology and an opportunity to define new more potent and specific therapeutic targets future.